Therapeutic use of tromethamine salt of L-ampicilline in hematopoiesis, immuno-oncology therapy, and regulation of lipoprotein and apolipoprotein levels

ABSTRACT

A method for increasing the number of CD4+ T-lymphocytes in the serum of a subject in need of such treatment comprising administering to the subject a pharmaceutical composition comprising an amount of an L-isomer of β-lactam effective to increase the number of CD4+ T-lymphocytes in said patient&#39;s serum.

TECHNICAL FIELD

This present invention relates to the use of levorotatory β-lactams inhematopoiesis, immune-oncology therapy, and the regulation oflipoprotein and apolipoprotein levels.

BACKGROUND OF THE INVENTION

Human leukocyte elastase (HLE, EC.3.4.21.37) is a serine proteinase thatis synthesized and processed as a single molecular protein that istargeted exclusively for the cell surface (HLE-CS) early in ontogeny oflymphoid and myeloid cells when they are too immature to form granules.However, HLE is targeted for granule compartmentalization (HLE-G) laterin ontogeny when cells develop the capability to form granules (Gullberget al., 1995; Garwicz et al., 2005). Whereas HLE-G has enzymaticactivity, HLE-CS acts as a receptor, and there is no evidence of itshaving enzymatic activity.

The primary physiologic mechanism for controlling the enzymatic activityof HLE-G is the abundant proteinase inhibitor alpha-1 proteinaseinhibitor (α1PI, α1 antitrypsin). When bound to HLE-G, a covalent-likecomplex is formed in which neither α1PI, nor HLE-G are cleaved(Dementiev et al., 2006). Similarly, when α1PI binds to HLE-CS, acomplex is formed which does not appear to involve completion ofenzymatic activity, and the complex induces polarization offunctionally-related receptors and cell motility (Wolf et al., 2003).Functionally-related receptors include the chemokine receptor CXCR4(CD184), CD4, and the T cell antigen receptor (TcR).

In healthy individuals, 98% of α1PI is in the active form. Normal rangesare 18-53 μM active and 0-11 μM inactive α1PI (Bristow et al., 2001).Active α1PI circulates in blood in two isoforms in dynamicequilibrium: 1) native α1PI, which binds irreversibly to HLE-CS, and 2)thiol-modified α1PI, which binds reversibly to HLE-CS (Tyagi, 1991).

Inactive α1PI arises during infection or inflammation via modificationof active α1PI by factors released from microorganisms or host cells.Inactive α1PI can arise by complexing with HLE-G or HLE-CS, beingcleaved by proteinases other than HLE, or oxygenation. In itsinactivated form, α1PI binds to low density lipoprotein (LDL), apoB, andmembers of the LDL receptor family (LDL-RFMs), whereby α1PI facilitatesLDL uptake into cells (Mashiba et al., 2001; Janciauskiene et al.,2001).

Binding of active α1PI to HLE-CS at the leading edge of migrating cellsinduces aggregation and polarization of LDL-RFMs with otherfunctionally-related receptors (Bristow et al., 2003; 2008; Bristow andFlood, 1993). Cellular locomotion repositions the HLE-CS complexincluding functionally-associated receptors to the trailing edge of thecell where LDL-RFMs on the same cell bind to the α1PI-HLE-CS complexesthrough an interaction that involves the α1PI C-terminal domain (C-36,VIRIP) (Kounnas et al., 1996; Janciauskiene el al., 2001). Thisinteraction induces internalization (endocytosis) of LDL-RFMs includingfunctionally-related receptors within the aggregate and entities boundto them such as lipoproteins and viruses. This action furthersretraction of the trailing edge of the migrating cell thereby promotingforward locomotion (Kounnas el al., 1996; Cao el al., 2006; Bristow etal., 2003; Bristow et al., 2013).

The recycling of endocytosed receptors and polarization at the leadingedge of a migrating cells followed by endocytosis at the trailing edgeoperates somewhat like a conveyor belt. If one of the componentsinvolved in this conveyor belt mechanism is missing or blocked, the cellhalts migrating. For example, bacteria, snake bites, blood clotting, andmost other non-normal situations produce non-normal proteases whichcleave sentinel proteinase inhibitors including α1PI. When α1PI isinactivated, it can no longer bind its receptor HLE-CS. In the absenceof α1PI-HLE-CS complexes, the LDL-RFMs are not triggered for endocytosisand this causes blood cells to stop migrating. This mechanistic processprovides a method for locomoting immune cells to sample the environmentfor nutrients (e.g. lipoproteins and insulin-coupled glucose), toxicmaterial (e.g. viruses, bacterial enzymes, inflammatory products), orinert material (e.g. recycled receptors). Due to the dynamics of theprocess, targeting HLE-CS using levorotatory β-lactams allows regulationof hematopoiesis, lipoprotein levels, and unwanted tissue degradation.

Cell motility results from selective and sequential adherence andrelease produced by activation and deactivation of receptors (Wright andMeyer, 1986; Ali et al., 1996), consequent polar segregation of relatedmembrane proteins to the leading edge or trailing uropod, and bothclockwise and counterclockwise propagation of Ca++ waves which initiatefrom different locations in the cell (Kindzelskii and Petty, 2003).Thus, several aspects of the complex process may be quantitated. Themost direct and most easily interpreted method for quantitating cellmotility is the enumeration of adherent cells in response to achemotactic agent such as α1PI.

Applicant's co-pending U.S. patent application Ser. No. 13/302,821 isdirected to a method for increasing the number of circulating CD4+T-lymphocytes in subjects receiving antiretroviral therapy comprisingadministering to a subject in need of such treatment an amount of activeα1PI effective to increase the number of circulating CD4+T-lymphocytesin the subject.

Applicant's co-pending Ser. No. 13/948,446 is directed to a method ofmodulating LDL levels, HDL levels, cholesterol levels, and triglyceridelevels in a subject comprising administering to the subject in need ofsuch treatment a pharmaceutical composition comprising a therapeuticallyeffective amount of active α1PI, thereby modulating the distribution ofLDL levels, HDL levels, cholesterol levels, and triglyceride levels inthe subject.

The hematopoietic role of α1PI has not been therapeutically exploitedthus far. Its use has been limited due to its high cost and shortsupply. Previous therapeutic applications of α1PI have been restrictedto augmentation for the purpose of ameliorating respiratory distresssuch as occurs in emphysema and chronic obstructive pulmonary disease(COPD) in patients diagnosed with inherited α1PI deficiency.Commercially, α1PI is available from a few sources includingPROLASTIN®-C, (Grifols Therapeutics Inc., Research Triangle Park, N.C.)and ZEMAIRA® (CSL Behring LLC, King of Prussia, Pa.) as acryoprecipitate preparation isolated from human plasma.

What is needed in the art are small molecules to act as surrogates forα1PI. The present invention provides such small molecule compoundsuseful in arresting degradative enzymatic activity for the treatment ofrespiratory distress such as occurs in emphysema and COPD, modulatinglipoprotein and apolipoprotein levels, and for regulating the number ofcirculating CD4+ or CD8+ T-lymphocytes (CD4/CD8 ratio) in subjects inneed of such treatment.

SUMMARY OF THE INVENTION

It has now been unexpectedly discovered that all 5 classes of β-lactamantibiotics, including L-ampicillin, bind to soluble granule-associatedelastase (HLE-G) and to cell surface elastase (HLE-CS), induce receptorpolarization and stimulate cell motility. As these are the biologicalactivities of α1PI, β-lactams can be used as a surrogate for α1PI inbinding to HLE-CS thereby modulating lipoprotein and apolipoproteinlevels and regulating the number of circulating CD4+ or CD8+T-lymphocytes (CD4/CD8 ratio) in human subjects, as well as arrestingdegradative enzymatic activity for the treatment of respiratory distresssuch as occurs in emphysema and chronic obstructive pulmonary disease(COPD). Further, some β-lactams such as L-ampicillin can be immuneenhancing whereas others such as Cephelaxin can be immune suppressing.Most drugs that target cells, including α1PI, exhibit a bell shapedcurve where a high dose or high affinity is as ineffective as a low doseor low affinity, and the optimal dose of each drug is somewhere inbetween.

In one embodiment, the present invention provides a method forincreasing the CD4/CD8 ratio by increasing the number of CD4+T-lymphocytes or decreasing the number of CD8+ lymphocytes in the serumof a subject in need of such treatment comprising, administering to thesubject a pharmaceutical composition comprising an amount of alevorotatory β-lactam such as L-ampicillin effective to increase theCD4/CD8 ratio in said patient's serum.

In another embodiment, the present invention provides a method fordecreasing the CD4/CD8 ratio by decreasing the number of CD4+T-lymphocytes or increasing the number of CD8+ lymphocytes in the serumof a subject in need of such treatment comprising, administering to thesubject a pharmaceutical composition comprising an amount of alevorotatory β-lactam such as L-cephalexin effective to decrease theCD4/CD8 ratio in said patient's serum.

In another embodiment, the method further comprises the step ofdetermining the CD4/CD8 ratio, the number of CD4+ T-lymphocytes and thenumber of CD8+ lymphocytes in said patient's serum.

In a further embodiment the β-lactam is selected from a group consistingof Cephems.

In a further embodiment, the β-lactam is selected from a groupconsisting of

Penams.

In a further embodiment, the β-lactam is selected from a groupconsisting of Monobactams.

In a further embodiment, the β-lactam is selected from a groupconsisting of

Penems.

In a further embodiment, the β-lactam is selected from a groupconsisting of Carbapenems.

In a still further embodiment, the subject is suffering from secondaryimmune deficiency, such as occurs in malnutrition or HIV-1 disease or incancer therapy.

In a still further embodiment, the subject is suffering from cancer andis being treated with immune checkpoint inhibitors.

In a still further embodiment, the subject is suffering from immunehyperactivation such as occurs in autoimmunity or graft-versus-hostdisease.

In another embodiment, the subject is suffering from inherited α1PIdeficiency.

In another embodiment, the CD4/CD8 ratio or the number of CD4+T-lymphocytes in the subject is undesirably low.

In another embodiment, the CD4/CD8 ratio or the number of CD4+ Tlymphocytes in the subject is undesirably high, requiring immunesuppression therapy.

In another embodiment, the subject is a patient suffering from a solidtumor including but not limited to melanoma, renal cell carcinoma,non-small cell lung cancer, bladder cancer, cervical cancer, gastriccancer, liver cancer, pancreatic cancer, and brain cancer.

In another embodiment, the subject is a patient exposed to environmentaltoxins such as radiation or chemotherapy.

In another embodiment, the subject is suffering from at least onecondition selected from the group consisting of viral infection,bacterial infection, and malnutrition.

In another embodiment, the subject is suffering from an autoimmunedisease.

In another embodiment, the subject is suffering from graft-versus-hostdisease.

In a further embodiment, the present invention provides a method formodulating LDL levels, HDL levels, cholesterol levels, and otherlipoprotein and apolipoprotein levels derived from or resulting fromdietary fats, LDL, HDL and cholesterol and other lipoproteins andapolipoproteins in a subject comprising administering to a subject inneed of such treatment a pharmaceutical composition comprising atherapeutically effective amount of a β-lactam thereby modulating LDLlevels, HDL levels, cholesterol levels, and the levels of otherlipoproteins and apolipoproteins derived from or resulting from dietaryfats, LDL, HDL, cholesterol, and other lipoproteins and apolipoproteinsin the subject.

In another embodiment, the method further comprises determining LDLlevels, HDL levels, cholesterol levels, and the levels of otherlipoproteins and apolipoproteins derived from or resulting from dietaryfats, LDL, HDL cholesterol, and other lipoproteins and apolipoproteinsin said subject's serum before administration of the β-lactam.

In another embodiment, the method further comprises determining LDLlevels, HDL levels, cholesterol levels, and the levels of otherlipoproteins and apolipoproteins resulting from or derived from dietaryfats, LDL, HDL, cholesterol, and other lipoproteins and apolipoproteinsin said subject's serum after administration of the β-Lactam.

In yet another embodiment, modulation comprises lowering LDL levels andthe levels of lipoproteins and apolipoproteins resulting from or derivedfrom LDL.

In another embodiment, modulation comprises increasing HDL levels andthe levels of lipoproteins and apolipoproteins resulting from or derivedfrom HDL.

In another embodiment, modulation comprises modulating apoB48 or apoB100levels and the levels of lipoproteins and apolipoproteins resulting fromor derived from apoB48 or apoB100.

In another embodiment, the subject is a human or a non-human animal.

In another embodiment, an LDL inhibitor or cholesterol lowering drug isselected from the group consisting of statins, PCSK9 inhibitors,fibrate, niacin, and bile acid sequestrant is administered to thesubject.

In a particularly preferred embodiment the β-lactam is an L-ampicillinsalt.

In another preferred embodiment the levels of LDL and the levels ofother lipoproteins and apolipoproteins resulting from or derived fromLDL are lowered.

In another preferred embodiment the levels of HDL and the levels ofother lipoproteins and apolipoproteins resulting from or derived fromHDL are increased.

In a still further embodiment the present invention provides a methodfor treating patients suffering from congenital α1PI deficiency andsuffering from COPD comprising administering to said patients aneffective amount of a β-lactam.

In yet another embodiment the present invention provides a compositionof matter comprising the tromethamine salt of L-ampicillin.

In another embodiment the present invention provides a pharmaceuticalcomposition comprising the tromethamine salt of L-ampicillin and apharmaceutically acceptable carrier, excipient, or diluent.

In a still further embodiment the present invention provides a methodfor treating patients suffering from congenital α1PI deficiency andsuffering from COPD comprising administering to said patients aneffective amount of a β-lactam.

In a further embodiment, the present invention provides a method fortreating a patient suffering from respiratory distress caused byemphysema and COPD comprising administering to a patient in need of suchtreatment of a therapeutically effective amount a β-Lactam.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thepresent description, drawings, and claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A-1C are graphs demonstrating that binding to HLE-CS inhibitschemokine-induced locomotion of human leukemic cells and enhanceslocomotion of human stem cells. FIG. 1A depicts adherent cellsenumerated in response to chemokines CXCL12α(●), CCL3 (▪), CCL4 (▴), andCCL5 (▾) or in response to proteinase inhibitors α1PI (▴), antithrombinIII (ATIII) (▪), and anti-chymotryspin (α₁ACT); FIG. 1B depicts that HIVpermissive (plus) clone 10 (●), but not HIV non-permissive (minus) clone17 (▪), was stimulated to adhere by MAAPVCK, a synthetic peptideinhibitor for HLE and by TPCK, a synthetic peptide inhibitor forchymotrypsin. Plus clone 10 (●), but not minus clone 17 (▪), wasstimulated to adhere in response to the HIV fusion peptide (FLGFL).Neither subclone was influenced by the thrombin agonist peptide(SFLLRN). Two different preparations of α₁PI were determined to be 32.7%(●) and 8.3% active (▪); FIG. 1C depicts Plus clone 10 stimulated bythese two α₁PI preparations responded to equivalent optimalconcentrations of active α₁PI, but different concentrations of inactiveα₁PI.

FIGS. 2A and 2B are photographs of Mueller-Hinton agar plates comparingthe antibiotic activity of D-ampicillin (2A) with the antibioticactivity of L-ampicillin (2B).

FIGS. 3A and 3B are photographs showing the stimulation of cellmigration and endocytosis induced by L-ampicillin.

FIG. 4 is a graph showing the normalized % change in LDL levels inDiet-induced obesity mice (DIO) mice treated pursuant to the presentinvention with vehicle, ezetimibe (10 mg/kg), L-ampicillin (50 mg/kg),or L-ampicillin (5 mg/kg).

FIG. 5 is a cDNA microarray analysis performed using peripheralmononuclear blood cells (PMBC) harvested from 1 uninfected, untreatedvolunteer and 2 HIV-1 infected individuals on ritonavir therapy; thegene expression ratio of HIV-1 infected to uninfected cells wascalculated

FIGS. 6A and 6B are graphs showing the changes in the number of Tlymphocytes (FIG. 6A) and T progenitor cells (FIG. 6B) following 2 weeksof treatment with L-ampicillin.

FIG. 7A are photographs of representative tumors treated withL-ampicillin or with anti-PD-1 or with L-ampicillin in combination withanti-PD-1. FIG. 7b are bar graphs showing the mean tumor weight withineach group determined as the difference between the left kidney weightand the right kidney weight in each mouse

FIG. 8A and FIG. 8B are diagrams showing the absolute configuration ofD-ampicillin (8A) and L-ampicillin (8B).

DETAILED DESCRIPTION Definitions

The term “about” or “approximately” usually means within an acceptableerror range for the type of value and method of measurement. Forexample, it can mean within 20%, more preferably within 10%, and mostpreferably still within 5% of a given value or range. Alternatively,especially in biological systems, the term “about” means within about alog (i.e., an order of magnitude) preferably within a factor of two of agiven value.

“Active α1PI” is the fraction of full length, unmodified α1PI in plasmaor other fluids that has the capacity to inhibit elastase activity.

“Inactive α1PI” is the fraction of α1PI in plasma or other fluids thatdoes not have the capacity to inhibit elastase activity. Active α1PI maybe inactivated by proteolytic cleavage, proteinase complexing, antibodycomplexing, or oxidation.

“β-lactam antibiotics” are defined herein as members of the groupconsisting of Cephalosporins (Cephems); Penicillins (Penams);Monobactams; Penems and Carbapenems.

“Substantially no bactericidal activity” as used herein in reference toL-ampicillin is defined by the 2014 Clinical & Laboratory StandardsInstitute (CLSI) criteria as a 4-fold dosage difference forampicillin-resistant vs susceptible E. coli (Table 2A of the 2014 CLSIM100-S24) (CLSI, 2014). Because the dosage difference betweenD-ampicillin and L-ampicillin is 10-fold, L-ampicillin is not consideredto be an effective antibiotic.

The terms “decrease”, “decreased”, “reduced”, “reduction” or“down-regulated” are all used herein generally to mean a decrease by astatistically significant amount. However, for avoidance of doubt,“reduced”, “reduction”, “down-regulated” “decreased” or “decrease” meansa decrease by at least 10% as compared to a reference level, for examplea decrease by at least about 20%, or at least about 30%, or at leastabout 40%, or at least about 50%, or at least about 60%, or at leastabout 70%, or at least about 80%, or at least about 90% or up to andincluding a 100% decrease (i.e. absent level as compared to a referencesample), or any decrease between 10-100% as compared to a referencelevel, or at least about a 0.5-fold, or at least about a 1.0-fold, or atleast about a 1.2-fold, or at least about a 1.5-fold, or at least abouta 2-fold, or at least about a 3-fold, or at least about a 4-fold, or atleast about a 5-fold or at least about a 10-fold decrease, or anydecrease between 1.0-fold and 10-fold or greater as compared to areference level.

The terms “increased”, “increase” or “up-regulated” are all used hereinto generally mean an increase by a statistically significant amount; forthe avoidance of any doubt, the terms “increased” or “increase” means anincrease of at least 10% as compared to a reference level, for examplean increase of at least about 20%, or at least about 30%, or at leastabout 40%, or at least about 50%, or at least about 60%, or at leastabout 70%, or at least about 80%, or at least about 90% or up to andincluding a 100% increase or any increase between 10-100% as compared toa reference level, or at least about a 0.5-fold, or at least about a1.0-fold, or at least about a 1.2-fold, or at least about a 1.5-fold, orat least about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 1.0-fold and 10-fold or greater as compared to areference level.

As used herein, “modulate” or “modulating” refers to increase ordecrease, or an increase or a decrease, for example an increase in thelevel of HDL or a decrease in the level of LDL or an increase in thenumber of immune cells, or a decrease in the number of immune cells.

Non-limiting examples of lipoproteins and apolipoproteins derived fromHDL, LDL and cholesterol include chylomicrons, lipoprotein(s),intermediate density lipoproteins (IDL), very low density lipoproteins(VLDL), and apolipoproteins (apo) including apolipoproteins A, B, C, D,E, H, and L, and their molecular variants including apoA-I, apoA-II,apoA-IV, apoA-V, apoB48, apoB100, apoC-I, apoC-II, apoC-III, andapoC-IV. Exchangeable apolipoproteins (apoA, apoC and apoE) have thesame genomic structure and are members of a multi-gene family thatevolved from a common ancestral gene. ApoA1 and ApoA4 are part of theAPOA1/C3/A4/A5 gene cluster on chromosome 11. Hundreds of geneticpolymorphisms of the apolipoproteins have been described, and many ofthem alter their structure and function as disclosed in Holmes et al,2011.

A “level”, in some embodiments, may itself be a relative level thatreflects a comparison of levels between two states. Relative levels thatreflect a comparison (e.g., ratio, difference, logarithmic difference,percentage change, etc.) between two states (e.g., healthy anddiseased). The use of relative levels is beneficial in some casesbecause, to an extent, they exclude measurement related variations(e.g., laboratory personnel, laboratories, measurements devices, reagentlots/preparations, assay kits, etc.). However, the invention is not solimited.

As used herein the terms “therapeutically effective” and “effectiveamount”, used interchangeably, apply to a dose or amount refer to aquantity of a composition, compound or pharmaceutical formulation thatis sufficient to result in a desired activity upon administration to ananimal in need thereof. Within the context of the present invention, theterm “therapeutically effective” refers to that quantity of acomposition, compound or pharmaceutical formulation that is sufficientto reduce or eliminate at least one symptom of a disease or conditionspecified herein. When a combination of active ingredients isadministered, the effective amount of the combination may or may notinclude amounts of each ingredient that would have been effective ifadministered individually. The dosage of the therapeutic formulationwill vary, depending upon the nature of the disease or condition, thepatient's medical history, the frequency of administration, the mannerof administration, the clearance of the agent from the host, and thelike. The initial dose may be larger, followed by smaller maintenancedoses. The dose may be administered, e.g., weekly, biweekly, daily,semi-weekly, etc., to maintain an effective dosage level.

Pharmaceutical compositions include an active agent, i.e., a β-lactamand a pharmaceutically acceptable carrier, excipient, or diluent.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the compound is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water or aqueoussaline solutions and aqueous dextrose and glycerol solutions arepreferably employed as carriers, particularly for injectable solutions.Alternatively, the carrier can be a solid dosage form carrier, includingbut not limited to one or more of a binder (for compressed pills), aglidant, an encapsulating agent, a flavorant, and a colorant. Suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” by E. W. Martin.

When formulated in a pharmaceutical composition, a therapeutic compoundof the present invention can be admixed with a pharmaceuticallyacceptable carrier or excipient. As used herein, the phrase“pharmaceutically acceptable” refers to molecular entities andcompositions that are generally believed to be physiologically tolerableand do not typically produce an allergic or similar untoward reaction,such as gastric upset, dizziness and the like, when administered to ahuman.

The term “pharmaceutically acceptable derivative” as used herein meansany pharmaceutically acceptable salt, solvate or prodrug, e.g. ester, ofa compound of the invention, which upon administration to the recipientis capable of providing (directly or indirectly) a compound of theinvention, or an active metabolite or residue thereof. Such derivativesare recognizable to those skilled in the art, without undueexperimentation. Nevertheless, reference is made to the teaching ofBurger's Medicinal Chemistry and Drug Discovery, 5th Edition, Vol 1:Principles and Practice, which is incorporated herein by reference tothe extent of teaching such derivatives. Preferred pharmaceuticallyacceptable derivatives are salts, solvates, esters, carbamates, andphosphate esters. Particularly preferred pharmaceutically acceptablederivatives are salts, solvates, and esters. Most preferredpharmaceutically acceptable derivatives are salts and esters.

All classes of β-lactams contain proton donating groups, e.g. carboxylicacid or hydrofluoric acid, allowing them to be easily transformed intopharmaceutically acceptable salts. Non-limiting examples ofpharmaceutically acceptable salts may be formed with cations includingbenzathine, calcium, cholinate, diethanolamine, diethylamine, lysine,magnesium, meglumine, piperazine, potassium, procaine, silver, sodium,tromethamine, or zinc. Further, nonlimiting examples of pharmaceuticallyacceptable salts may be formed with anions including acetate, benzoate,besylate, bromide, camphorsulfonate, chloride, chlortheophyllinate,citrate, ethandisulfonate, fumarate, gluceptate, gluconate, glucuronate,hippurate, iodide, isethionate, lactate, lactobionate, laurylsulfate,malate, maleate, mesylate, methylsulfate, naphthoate, napsylate,nitrate, octadecenoate, oleate, oxalate, pamoate, phosphate,polygalacturonate, succinate, sulfate, sulfosalicylate, tartrate,tosylate, or trifluoroacetate (Paulekuhn et al., 2007).

While it is possible to use a composition provided by the presentinvention for therapy as is, it may be preferable to administer it in apharmaceutical formulation, e.g., in admixture with a suitablepharmaceutical excipient, diluent, or carrier selected with regard tothe intended route of administration and standard pharmaceuticalpractice. Accordingly, in one aspect, the present invention provides apharmaceutical composition or formulation comprising at least one activecomposition, or a pharmaceutically acceptable derivative thereof, inassociation with a pharmaceutically acceptable excipient, diluent,and/or carrier. The excipient, diluent and/or carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof.

In a particularly preferred embodiment the β-lactam is the tromethaminesalt of L-ampicillin prepared using tromethamine(hydroxymethyl)aminomethane, CAS no. 77-86-1) and producing(tromethamine; (2 S,5R,6R)-6-{[(2S)-2-Amino-2-phenylacetyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylicacid). L-ampicillin tromethamine salt is a diastereomer of D-ampicillinsodium salt (sodium;(2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate).While L-ampicillin tromethamine salts have not been reported in thescientific literature, their structure has been confirmed by theinventors by nuclear magnetic resonance proton and carbon analysis usinga Bruker Ascend™ 700 MHz spectrometer.

Because L-ampicillin and D-ampicillin are produced synthetically orsemi-synthetically using chiral starting materials, they are not foundas racemic mixtures (Example 6). L-ampicillin is soluble in tromethamineat pH 8.6 and is not soluble in 100% denatured ethanol whereasD-ampicillin is poorly soluble in tromethamine, at pH 8.6 and is solubleat 6133 g/L water as well as in 100% denatured ethanol (demonstratedexperimentally by the inventors and disclosed in Bartzatt et al., 2007).

The compositions or pharmaceutical formulations of the invention can beformulated for administration in any convenient way for use in human orveterinary medicine.

For human therapy, the pharmaceutical formulations or compositions,including each of the active agents, are prepared in accordance withgood manufacturing process (GMP) standards, as set by the Food & DrugAdministration (FDA). Quality assurance (QA) and quality control (QC)standards will include testing for purity and function and otherstandard measures.

Although there is extensive knowledge about treating inflammation andregulating the reactivity of the immune system, for patients withsecondary immune deficiency there are only 2 FDA approved therapeuticoptions, gamma globulin infusion and bone marrow transplantation. Bothof these options are painful, have serious safety issues, and offerlimited efficacy. Every patient with cancer-treatment induced immunedeficiency, every HIV-infected patient, every patient with other virus-or environmentally-induced immune deficiency, suffers because of thelack of a therapeutic agent to treat secondary immune deficiency.

The present inventors previously discovered that the human protein α1PIis both safe and effective in restoring the immune system in patientswith secondary immune deficiency by binding to its cell surfacereceptor, HLE-CS. While the use of α1PI for treating the large number ofpatients with secondary immune deficiency is not economically feasibledue to supply and cost issues, synthetic drugs that mimic the biologicalactivity of α1PI by binding to HLE-CS are needed in the art.

Disclosed herein is surprising and unexpected discovery that L-isomersof (3-lactams, including the salts of the L-isomer of D-ampicillin(hereinafter “L-ampicillin”), which have substantially no bactericidalactivity (as defined herein), have the biological properties of activeα1PI required for their use as surrogates for α1PI for the purposes of:

(1) Ameliorating respiratory distress such as occurs in emphysema andchronic obstructive pulmonary disease (COPD) in patients suffering fromcongenital α1PI deficiency;

(2) Treating patients suffering from immune dysfunction by inducingmobilization of lymphoid-committed progenitor cells from hematopoietictissue. This produces elevated levels of circulating T-lymphocytes inindividuals in need of such treatment due to cancer, atherosclerosis,autoimmunity, stem cell transplantation, organ transplantation, HIV-1infection, microbial infection, leukemia, and other diseases affected bycells of the immune system.

(3) Treating patients suffering from hyperlipidemia by regulatinglipoprotein and apolipoprotein levels, for example, HDL and LDL andcholesterol levels and other lipoproteins and apolipoproteins derivedfrom dietary fats, HDL and LDL and cholesterol as a consequence of itsability to elevate T-lymphocytes which transport lipoproteins andapolipoproteins throughout the tissues.

(4) Treating cancer patients suffering from tumors.

(5) Treating cancer patients suffering from tumors in combination withimmune checkpoint inhibitors.

It is surprising and unexpected that small molecules such aslevorotatory β-lactams have the biological properties of active α1PI andcan be used as surrogates in the above-described methods.

L-ampicillin and salts thereof are particularly preferred for use in thepresent invention because as shown below in Example 3, L-ampicillin hassubstantially no anti-bactericidal activity as defined herein.

Functional Capacity of L-Ampicillin and Other Levorotatory β-Lactams ofthe Present Invention

Pursuant to the present invention, L-ampicillin ((2S, 5R,6R)-6-((S)-2-amino-2 phenylacetamcido)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-acid, CAS#19379-33-0) and other β-lactams can be used as surrogates for α1PI inthe methods described herein. The present inventors discovered thatL-ampicillin binds to soluble and cell surface elastase and modulateslipoprotein and apolipoprotein levels, induces receptor polarization,stimulates cell motility, and increases the number of CD4+ T-lymphocytesin the serum of a subject in need of such treatment.

Based on these properties, L-ampicillin and other levorotatory β-lactamscan be used in a method for regulating the number of CD4+ T-lymphocytesin the serum of a subject in need of such treatment, modulating LDLlevels, HDL levels, cholesterol levels, and the levels of otherlipoproteins and apolipoproteins resulting from or derived from LDL,HDL, cholesterol, and other lipoproteins and apolipoproteins in asubject including patients suffering from congenital α1PI deficiency.

As set forth in Example 2 below, β-lactams bind to and inactivatesoluble elastase:

The procedures for measuring the capacity of substances to inhibitsoluble forms of elastase or HLE-G are well known to those of ordinaryskill in the art (U.S. Pat. No. 6,887,678; Bristow et al., 1998).Briefly, soluble human leukocyte elastase (HLE-G) is incubated for 2minutes with a test substance, and to this mixture is added the elastasesubstrate succinic-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA,Sigma-Aldrich). Results are detected by measuring the color change at405 nm. IC50 is calculated from these results.

As set forth in Example 4 below, L-ampicillin induces receptorpolarization and stimulates cell motility.

The procedures for inducing receptor polarization have been described(Bristow et al., 2003). The cells of interest (monocytes, lymphocytes,neutrophils, or other blood cells, e.g. leukemic cells) are isolatedfrom blood or tissue using standard techniques (for example, asdisclosed in Messmer et al., 2002) and examined for reactivity withL-ampicillin.

To examine receptor polarization, microscope slides are prepared byadding serial dilutions of a β-lactams. Cells are added to themicroscope slides and incubated for 30 minutes in humidified 5% CO₂ at37° C. Unattached cells are removed by washing, and attached cells arefixed by application of 4% paraformaldehyde after which attached cellsare counted by light microscopy and photographed using confocalmicroscopy

As demonstrated in Example 4 below, L-ampicillin stimulates cellmotility. Confocal microscopy was used to demonstrate that the cellstreated with L-ampicillin exhibited the morphology of cells undergoingcell motility.

To demonstrate that L-ampicillin mobilizes lymphoid-committed progenitorcells, the Jackson Laboratory C57BL/6 Diet-Induced Obesity (DIO)diabetic mouse model is used. The DIO mouse model is used to assess thecapacity of L-ampicillin to mobilize lymphoid- or myeloid-lineage cellsand lower LDL levels.

Therapeutically Effective Amounts of Levorotatory β-Lactams for Use inthe Present Invention

According to the manufacturer, the recommended regimen for PROLASTIN-C®(human α1PI) for treating α1PI deficiency is repeated weekly infusionsof 60 mg/kg at a rate of 0.08 ml/kg/minute. The specific activity ofPROLASTIN-C® is 70%, wherein specific activity is defined as theinhibition of elastase activity as described in the package insert.Thus, the recommended dose of α1PI is 42 mg/kg of active α1PI to achievehalf the normal level of α1PI and 84 mg/kg or 1.53 millimol/kg toachieve a normal level of α1PI. Since L-ampicillin has a mass of 349.41mg/mole, 1.53 millimol/kg is 0.53 mg/kg is the target dose ofL-ampicillin. By comparison, the pediatric dose of D-ampicillin fortreating bacterial infections is 50-100 mg/kg/day (every 6 hr.), andpediatric blood volume is 70 ml/kg; thus the pediatric dose ofD-ampicillin is 50-100 mg/70 ml which is equivalent to 2-4 millimol/dayor 0.7-1.3 millimol every 6 hr (q6 hr). The commonly used adult dose is750-1500 mg/day (q6 hr), and adult blood volume is 5 L which isequivalent to 0.4-0.9 millimol/day or 0.2-0.3 millimol q6 hr. Thus, thecommonly used doses of D-ampicillin are approximately equivalent to thetherapeutically effective doses of L-ampicillin. Whereas α1PI treatmentis given weekly by infusion, L-ampicillin can be administered orally andmore frequently, if necessary.

In one preferred embodiment, the therapeutically effective amounts oflevorotatory β-lactams for use in this embodiment of the presentinvention will be between about 100 mg and about 3000 mg/kg body weightadministered 4 times per day (qid) for adults and between about 10 mgand 200 mg/kg qid for pediatrics.

The preferred route of administration for levorotatory β-lactams is oralbut other routes, such as subcutaneous injection, intramuscularinjection, and topical administration can be used.

L-ampicillin for use in the present invention is commercially availablefrom multiple sources including BOC Sciences, Shirley, N.Y. L-ampicillinand the salt of L-ampicillin can be chemically synthesized using, forexample, the method described in Example 6 below. Commercial sources forother β-lactams for use in the present invention are set forth inExample 9 below.

Treatment Outcome Measurements:

To determine whether treatment affects soluble elastase inhibitoryactivity, individuals are monitored weekly for changes in the active andinactive α1PI blood levels (Bristow et al., 1998) (U.S. Pat. No.6,887,678). Briefly, a constant amount of active site-titrated elastaseis allowed to incubate with serial dilutions of serum for 2 minutes at37° C. after which an elastase substrate is added. Determination of themolecules of substrate cleaved by residual, uninhibited elastase is usedto calculate the molecules of active and inactive α1PI in blood. Changesin measurements of active and inactive α1PI activity are followed upwith determination of whether the changes are due to physiologicalchanges or interference in measuring active and inactive α1PI due to thepresence of the β-lactam in blood. For patients receiving β-lactamtreatment, active and inactive α1PI are measured before, during, andafter treatment.

To determine the effectiveness of treatment on inducing changes inlevels of targeted blood cell populations, treated individuals aremonitored weekly for changes in complete blood count and differential,as well as for changes in specific subsets of blood cells such as CD4+lymphocyte cells and HLE-CS+ cells using flow cytometry (Bristow et al.,2001; Bristow, 2001; U.S. Pat. No. 6,858,400). Briefly, 100 μl of wholeblood is incubated with a panel of fluorescently-labeled monoclonalantibodies approved by the FDA for medical diagnostics (e.g.,commercially available from BD Diagnostics, Franklin Lakes, N.J.). Theseantibodies are selected to specifically recognize the cell receptorsthat uniquely identify the cell population of interest. Identificationand enumeration of the cells in blood that are bound to the monoclonalantibodies is performed using flow cytometry.

Levorotatory β-lactams can also be used in a method for modulating LDLlevels, HDL levels, cholesterol levels and the levels of otherlipoproteins and apolipoproteins resulting from or derived from LDL, HDLand cholesterol such as apoA, apoB, apoC, and apoE in a subjectcomprising administering to a subject in need of such treatment apharmaceutical composition comprising a therapeutically effective amountof a β-lactam; thereby modulating LDL levels, HDL levels and cholesterollevels and the levels of other lipoproteins and apolipoproteinsresulting from or derived from LDL, HDL, cholesterol and otherlipoproteins and apolipoproteins in the subject.

In one preferred embodiment, the levels of LDL and the levels of otherlipoproteins and apolipoproteins resulting from or derived from LDL arelowered.

In another preferred embodiment, the levels of HDL and the levels ofother lipoproteins and apolipoproteins resulting from or derived fromHDL are increased.

Levorotatory β-lactams can also be used in a method for regulatinghematopoiesis to modulate the number of CD4+ T cells and CD8+ T cells ina subject comprising administering to a subject in need of suchtreatment a pharmaceutical composition comprising a therapeuticallyeffective amount of a β-lactam; thereby modulating the number of CD4+CD8+ progenitor T cells and the resulting number of CD4+ T cells andCD8+ T cells.

In a still further embodiment, the present invention provides a methodfor treating patients suffering from congenital α1PI deficiency andsuffering from COPD comprising administering to said patients aneffective amount of a β-lactam.

In yet another embodiment, the present invention provides a compositionof matter comprising the tromethamine salt of L-ampicillin.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising the tromethamine salt of L-ampicillin and apharmaceutically acceptable carrier, excipient, or diluent.

The present invention is described below in examples which are intendedto further describe the invention without limiting the scope thereof.

Example 1: α1PI Inhibits SDF-1 Induced Migration of Human Leukemic Cellsand Enhances Migration of Human Stem Cells

Human acute myeloid leukemia cells (AML) not only secrete HLE-G, butalso express HLE-CS constitutively on the cell surface in a manner thatis regulated by the CXCR4/SDF-1 axis (Tavor et al., 2005).Pre-incubation of AML cells with α1PI significantly reduced their SDF-1dependent migration in all AML cells tested using an in vitro transwellassay (Tavor et al., 2005). Further, in a mouse model it was found thatα1PI inhibited homing of transplanted human stem cells to bone marrowand egress of transplanted AML cells from bone marrow (Lapidot andPetit, 2002).). The influence was shown to occur by the action of α1PIon HLE-CS (Bristow et al., 2008; Bristow et al., 2012).

When AML cells were treated with α1PI, SDF-1-induced pseudopodiaformation was prevented. These results are in contrast to previousstudies using a U937 promonocytic cell line which demonstrated thatα1PI-induced pseudopodia formation and inhibition of cell migration wasprevented by pretreatment with SDF-1 (FIG. 1) (Bristow et al., 2003).This difference was resolved by examining the kinetics of influence ofα1PI on cells and emphasizes the importance of α1PI and SDF-1 inpromoting cell migration of various cells depending on their stage ofdifferentiation and order of interaction with cells (Bristow et al.,2013).

For the adherence assay, sterile coverslips (12 mm diameter, Sigma) werewashed in endotoxin-free water and prepared by delivering a 10 μl volumecontaining various dilutions of one of the following stimulants in HBSSwithout calcium and magnesium: CCL3 (MIP-1α, Peprotech, Inc., RockyHill, N.J.), α1PI CCL4, (MIP-1β, Peprotech), CCL5, (RANTES, Peprotech),CXCL12, (SDF-1, Peprotech), (Cat. # A6150, lot #82H9323, Sigma), α₁PI(A9024, lot #115H9320, Sigma), α₁ antichymotrypsin (α₁ACT, Calbiochem,La Jolla, Calif.), antithrombin III (ATIII (Sigma), C1 esteraseinhibitor (Clinh, Calbiochem),methoxysuccinyl-L-Ala-L-Ala-L-Pro-L-Val-chloromethylketone (MAAPVCK,Sigma), N-tosyl-L-phenylalanine chloromethylketone (TPCK, Sigma), asynthetic peptide representative of the thrombin agonist (SFLLRN,Ser-Phe-Leu-Leu-Arg-Asn), or a synthetic peptide representative of theHIV fusion domain solubilized in 10% EtOH (FLGFL, Phe-Leu-Gly-Phe-Leu).

To coverslips prepared with chemoattractants as described above, 10⁶cells in 90 μl HBSS were mixed to uniformity, and incubated for 30 minin humidified 5% CO₂ at 37° C. without dehydration. To detectinteracting effects of stimulants, cells were delivered in an 80 μlvolume to coverslips previously prepared with 10 μl of one stimulant,mixed to uniformity, and incubated for 15 min at 37° C. Subsequently, anadditional stimulant was delivered in a 10 μl volume to each coverslip,mixed with pre-incubated cells to uniformity, and incubated for 30-60min at 37° C. After stringently washing coverslips free of non-adherentcells, adherent cells were fixed by incubation for 10 min at 20° C. with4% paraformaldehyde in PBS containing 2.5 μM of the nuclear staining,acridine orange. Slides were examined by epi-illumination UV microscopyon a Zeiss Axioskop Means and standard deviations were determined bycounting adherent cells in at least three fields/coverslip.

In FIG. 1 is shown that (A) Adherent cells were enumerated in responseto chemokines CXCL12α (●), CCL3 (▪), CCL4 (▴), and CCL5 (▾) or inresponse to proteinase inhibitors α₁PI (▴), ATIII (▪), and α₁ACT (●).Means and standard deviations represent the difference betweenstimulated and unstimulated adherence. (B) Plus clone 10 (●), but notminus clone 17 (▪), was stimulated to adhere by MAAPVCK, a syntheticpeptide inhibitor for HLE and by TPCK, a synthetic peptide inhibitor forchymotrypsin. Plus clone 10 (●), but not minus clone 17 (▪), wasstimulated to adhere in response to the HIV fusion peptide (FLGFL). TheHIV fusion peptide FLGFL was solubilized in 10% EtOH, and unstimulatedadherence of cells incubated with 10% EtOH in the absence of peptide was36±6 cells/field. Neither subclone was influenced by the thrombinagonist peptide (SFLLRN). Two different preparations of α₁PI weredetermined to be 32.7% (●) and 8.3% active (▪). (C) Plus clone 10stimulated by these two α₁PI preparations responded to equivalentoptimal concentrations of active α₁PI, but different concentrations ofinactive α₁PI. Adherence was optimized for each chemoattractant andadherence induced by each chemoattractant was enumerated in more thanthree separate experiments.

Examination of adherence stimulated by various agonists at various timepoints over a 24-hour period showed that optimal effects could bedetected between 30-60 min. Adherence of clones in independentexperiments using identical conditions did not vary. Unstimulatedadherence of U937 sub clones between independent experiments could beexplained in entirety by the bovine serum in which cells had beencultured, suggesting cells were conditioned by unknown serum componentsas previously demonstrated (Bristow et al., 2001; Bristow et al., 2003).

Example 2: Screening of β-Lactams for Activity; Inhibition of ElastaseProteinase Activity (HLE-G) and Stimulation of Cellular Adherence(HLE-CS)

Compounds were screened for 50% inhibitory activity (IC50) vs 75 μMsoluble, granule-associated human leukocyte elastase (HLE-G). As shownin Table 1, α1PI exhibited an IC50 of 38 μM, half the concentration ofHLE-G consistent with their known equimolar relationship. Cephems,Penams, Monobactams, Penems, and Carbapenems exhibited IC50 averagevalues of 219±48 μM, suggesting that β-lactams are effective for bindingto HLE at a molar excess of compound to HLE-G at IC50. As shown in Table1 below, all 5 classes of β-lactams bind to HLE-G and HLE-CS.

TABLE 1 All 5 classes of β-lactams bind to HLE-G and HLE-CS OptimalAdherence Compound Molar Adherent Concentration Class Compound IC50(μM)* Excess** Cells (nM)*** Cephems Cephalexin 134.1 1.8  27 ± 10 10Cefuroxine 166.7 2.2 34 ± 8 1 Penams D-Ampicillin 280.7 3.7 47 ± 8 1 PenV 262.0 3.5 30 ± 4 10 Dicloxacillin 231.6 3.1 26 ± 9 1 Amoxicillin 253.13.4  54 ± 20 1 L-Ampicillin 187.7 2.5 39 ± 7 1 Monobactams Aztreonam234.1 3.1 33 ± 4 1 Ezetimibe 276.7 3.7 40 ± 3 100 Penems Faropenem 203.12.7 23 ± 4 10 Carbapenems Doripenem 177.8 2.4 52 ± 6 100 *IC50 ofcompound versus HLE-G (75 μM). For comparison, α1PI is 38 μM at IC50.**Molar excess of compound to HLE-G at IC50. ***To each compound-treatedwell was added 2,000 U937 cells. For comparison (see FIG. 1C), theoptimal concentration of α1PI is 0.5 nm per 10,000 U937 cells yielding75 ± 19 adherent cells.

Example 3: L-Ampicillin has Substantially No Antibiotic Activity

To screen for antibiotic activity, the Clinical Laboratory StandardsInstitute (CLSI) approved protocol for the Kirby-Bauer Disk Test wasperformed to compare the antibiotic activities of D-ampicillin andL-ampicillin.

D-ampicillin-sensitive E. coli DH5-Alpha was cultured overnight in LBbroth, and the cell concentration was calibrated using McFarlandTurbidity Standard 0.05 for <300×10⁶ CFU. At this concentration ofcells, bacteria were spread on Mueller-Hinton agar plates. Filter disks(6 mm diameter) were placed on the agar and to each disk was applied 10μl of D-ampicillin or L-ampicillin in 10-fold serial dilutions beginningwith 50 mM concentration. After incubation for 16 hours at 37° C.,plates were examined for zones of inhibition. The results are shown inFIGS. 2A and 2B.

In FIG. 2A the diameters of the zones of inhibition of 5 mM and 0.5 mMD-ampicillin (3 cm at 10 o'clock and 1.5 cm at 8 o'clock, respectively)were equivalent to the diameters of the zones of inhibition of 50 mM and5 mM L-isomer (2.8 cm at 12 o'clock and 1.4 cm at 10 o'clock,respectively) in FIG. 2B. These results demonstrate that the L-isomerhas 10-fold less antibiotic activity compared to D-ampicillin.

The pediatric dose of D-ampicillin is 50-100 mg/kg/day (every 6 hr.),and pediatric blood volume is 70 ml/kg, thus the pediatric dose ofD-ampicillin is 50-100 mg/70 ml which is equivalent to 2-4 mM/day or0.7-1.3 mM every 6 hr (q6 hr). The commonly used adult dose is 750-1500mg/day (q6 hr), and adult blood volume is 5 L which is equivalent to0.4-0.9 mM/day or 0.2-0.3 mM q6 hr. D-ampicillin exhibits activity inthe Kirby-Bauer Disk Test at 0.5 mM which is within the range of thepediatric dose and exceeds the adult dose. However, L-ampicillin showsequivalent activity at 10-fold higher concentration (5 mM) and isineffective as an antibiotic at 0.5 mM concentration demonstrating thatit is ineffective as an antibiotic if used at this dosage. The 2014Clinical & Laboratory Standards Institute (CLSI) criteria forampicillin-resistant vs susceptible E. coli is a 4-fold dosagedifference. Because the dosage difference between D-ampicillin andL-ampicillin is 10-fold, L-ampicillin is not an effective antibiotic(Table 2A, 2014 CLSI M100-S24) (CLSI, 2014).

Example 4: Stimulation of Cell Migration and Endocytosis by L-Ampicillin

Receptor Polarization: U937 Clone 10 cells were cultured overnight inAIM-V serum-free medium. Cells were pelleted, re-suspended in AIM-V at2×10⁶/ml, and 250 μl (5×10⁵ cells) was added to Eppendorf tubespre-coated to prevent attachment. Cells were preconditioned with apositive control (α1PI, either PROLASTIN®-C or ZEMIRA®), D-ampicillin,L-ampicillin, or negative control (AIM-V medium) for 15 min at 37° C.,5% CO₂ to induce polarization of functionally-related plasma-membranereceptors including cell surface human leukocyte elastase (HLE-CS),CD4+, CXCR4, T cell antigen receptor (TcR), and the very low densitylipoprotein receptor (VLDLR) as previously shown (Bristow, et al.,2013).

Binding and Endocytosis: AT 2 chemically inactivated SHIV preparations,consisting of non-infectious virus with conformationally andfunctionally intact envelope glycoproteins, were provided by the AIDSVaccine Program (SAIC Frederick, Frederick, Md.). Cells were pulsed withvirus (30 ng p27 or p24 per 10⁶ cells) for 2 hr. at 2° C. which allowsbinding, but prevents endocytosis. Alternatively, cells were pulsed withvirus for 2 hr. at 37° C. which allows binding and endocytosis.Following pulsing for 2 hr., cells were mounted on Alcian blue slidesfor microscopy. The presence of inactivated virus in test cells wasdetected using dodecameric human CD4+-IgG1 provided by the Laboratory ofImmunoregulation, NIAID, NIH. This reagent specifically recognizesconformationally intact HIV 1/SIV envelope gp120. CD4-IgG1 was detectedusing HRP-conjugated Rb anti-human IgG (Sigma). CD4+ IgG-labeled cellswere coupled to Oregon 488 fluorochrome using the tyramide signalamplification system (Life Science Products, Boston, Mass., USA). Insome cases, cells stained on slides were permeabilized using 0.05%saponin during the blocking step and further stained with the nuclearstaining dye, 4′, 6-diamidino-2-phenylindole (DAPI), mounted, andexamined using epifluorescence microscopy using a Zeiss Axioplan or byconfocal microscopy using a Perkin Elmer Operetta High Content ImagingSystem. Cells were analyzed using 2 μm scanning from 3B.

Confocal images of cells preconditioned with L-ampicillin were captured6 μm above the attached surface of the cells.

The results are shown in FIGS. 3A and 3B. In FIG. 3A, cells maintainedat 2° C. exhibited receptor polarization. SHIV was detected only on theplasma membrane of polarized cells, never internal to the cells. Cellswere rounded and exhibited no evidence of cell migration. In FIG. 3B,cells maintained at 37° C. exhibited an extending leading edge andretracting trailing edge characteristic of cell migration. SHIV wasdetected internal to the cells, prominently along tubular structures atthe leading edges and in endosomes at the trailing edges of migratingcells. There was no SHIV binding in the presence of buffer alone (notdepicted). DAPI represents nuclei. Bar represents 25 μm, and arrowdepicts direction of locomotion of migrating cells.

Example 5: L-Ampicillin Lowers LDL Levels

The well-known Jackson Laboratory (Bar Harbor, Me.) C57BL/6 Diet-InducedObesity (DIO) mouse model represents human metabolic syndrome andelevated LDL levels. Mice are fed a high fat diet (60% fat) or normaldiet (10% fat) for various periods of time and lipoprotein levels weremeasured. As compared to the 10% DIO, the 60% DIO have significantlyelevated total cholesterol, HDL and LDL levels (p<0.001), but nottriglyceride levels (Tg) (FIG. 4).

Mice received daily treatment with ezetimibe (10 mg/kg) or L-ampicillin(50 mg/kg or 5 mg/kg) by oral gavage.

In accordance with a protocol used in a recently conducted humanclinical trial (NCT01731691), mouse peripheral blood was collected intoblood collection tubes, and serum was analyzed for lipoprotein levelsusing Beckman Coulter AU680 Chemistry System. In addition, cells wereanalyzed by flow cytometry to quantitate lymphocyte subsets includingCD3, CD4, and CD8. As compared to baseline levels (before treatment),lipoprotein levels (including, but not limited to total cholesterol,HDL, LDL, apoA, apoB, apoC, apoE) and blood cells expressing the abovementioned cellular markers were quantitated to determine changes due totreatment.

Diet-induced obesity mice (DIO, Jackson Laboratory) were fed a 60% fatdiet for 17 weeks. Twelve mice were assigned to each of 4 arms of thestudy including a vehicle control (Group 1), ezetimibe (Zetia, CaymanChemical), 10 mg/kg, (Group 2), L-ampicillin (50 mg/kg), (Group 3), andL-ampicillin (5 mg/kg, (Group 4). The compounds were delivered by oralgavage 5 days per week. Serum samples were collected on weeks 3, 6 and 8and were analyzed for levels of glucose, total cholesterol,triglycerides, HDL, LDL, and non-esterified fatty acids.

Because mice were maintained on the DIO diet throughout the study, bodyweight and LDL levels increased in the vehicle control (Group 1). Tocompare the effectiveness of compounds, mean values within eachtreatment group were normalized by forming a ratio to mean values ofvehicle, and the ratio was expressed as normalized % change using to theformula:100−(Treatment mean/Vehicle mean*100).

The results are shown in FIG. 4. At week 6, the normalized mean % changefor L-ampicillin, 5 mg/kg (Group 4, black bars) was −41.0±20.5% and forezetimibe (Group 2, grey bars) was −43.8±29.6%, significantly lower(P=0.001) than vehicle (Group 1, hatched bars) or L-ampicillin, 50 mg/kg(Group 3, white bars) (10% and 3.7%, respectively).

Example 6: Chemical Synthesis of L-Ampicillin

The step by step chemical synthesis of the compound is described indetail.

Procedure:

Compound 1 (phenylamine carboxylic acid is synthesized from toluene. Toa solution of compound 1 (500 mg, 3.31 mmol) (Wuxi Biortus Biosciences,Jiangyin, Jiangsu, P. R. China) in 2N sodium hydroxide (1.65 ml, 3.31mmol) stirred at 0° C., benzyl carbonochloridate (512 μL, 3.64 mmol) and2N sodium hydroxide (1.82 ml, 3.64 mmol) were simultaneously addeddropwise from two different syringes.

The reaction was stirred at RT for 45 minutes and a precipitateappeared.

Water was added and the solution was extracted with Et₂O. The aqueousphase was acidified with 1 N HCl and the desired product was extractedagain with Et₂O. The combined organic phases were dried over Na₂SO₄,filtered and evaporated to obtain title product (855 mg, 91% yield) as awhite solid.

Procedure:

Ethyl chloroformate (247 mg, 2.3 mmol) was added to an ice cold solutionof compound 2 (500 mg, 1.75 mmol), and triethylamine (231 mg, 2.3mmoles) in dry acetone (20 mL).

The mixture was stirred at 0° C. for 10 minutes.

Then the suspension was cooled to −50° C. and stirred vigorously duringaddition as rapidly as possible of an ice-cold solution of6-aminopenicillinic acid (417 mg, 1.93 mmol) in 3 percent sodiumbicarbonate.

The reaction mixture was stirred at 0° C. for one hour. The reactionmixture was allowed to reach room temperature over a period of 30minutes. Then the reaction mixture was concentrated under vacuum andthen washed with ether (3×100 mL). The aqueous layer was acidified to pH2 using ice cold dilute HCl and quickly extracted with ether. Thereaction mixture was concentrated under vacuum. The residue was purifiedby silica gel column chromatography with Hex/EtOAc (1/1) to provide thetitle product (400 mg, 35% yield) as a white solid.

Procedure:

To a solution of compound 3 (1.4 g, 2.9 mmol) in 100 mL of THF and 80 mLof water was added 1.4 g. of 20 percent Pd(OH)₂/C

The mixture was shaken under an atmosphere of hydrogen at a pressure of50 psi for 1.5 hours.

At this point the catalyst was removed by filtration and 1 g of freshcatalyst was added.

The mixture was shaken under hydrogen at 50 psi for a further 1 hour.

The catalyst was removed by filtration with celite and the bulk of theTHF was removed by evaporation in vacuo.

The pH of the residual aqueous phase was adjusted to 5.0 using AcOH andthe acidified solution was extracted with ethyl acetate (3×50 mL).

The aqueous phase was lyophilized.

250 m (24.71% yield) of the white solid was obtained.

Procedure:

To compound 4 was added 1M C₄H₁₁NO₃+0.22M HCl (1 g/30 mL) followed by1.8 L saline producing an orally available solution (53 mM). Compound 5is the tromethamine salt of L-ampicillin.

Example 7: α1PI in Hematopoiesis in Normal, Healthy Individuals

It has been previously demonstrated that in HIV-1 uninfected individualsCD4+ T lymphocyte counts (hereinafter “CD4 counts) are regulated by thenumber of cells expressing cell surface human leukocyte elastase(HLE-CS, active α1PI levels, and the number of cells expressing thechemokine receptor CXCR4 (r2=0.92, p, 0.001, n=31; Table 1 of referencedpublication (Bristow et al., 2012). In HIV-1 infected patients, activeα1PI becomes deficient due to disease processes, a situation in whichactive α1PI becomes rate limiting for CD4+ counts (Table 1 of referencedpublication (Bristow et al., 2001). The present inventors demonstratedthat active α1PI is strongly correlated with CD4+ counts in HIV-1infected individuals (r2=0.927, p<0.0001, n=26; FIG. 1 of referencedpublication (4).

In a clinical trial, it was demonstrated that therapeutic α1PIadministration caused an increase in CD4+ counts in HIV-1-infectedpatients with acquired α1PI deficiency due to HIV-1 infection and inHIV-1 uninfected patients with inherited α1PI deficiency. This confirmsthat α1PI regulates CD4+ counts in the presence or absence of HIV-1disease (FIG. 1 of referenced publication (Bristow et al., 2010)). Itwas demonstrated that the mechanism by which α1PI regulates CD4+ countsis by inducing the migration of cells through tissue, specificallythrough the thymus where CD4+ T-lymphocytes are generated. The bindingof circulating α1PI to T-lymphocytes that express HLE-CS (the receptorthat binds to active α1PI) and receptors for very low densitylipoprotein (VLDLR, the receptor that binds to inactive α1PI) results inconformational changes that promote the binding of the α1PI-HLE-CScomplex to VLDLR on the same cells, a situation that induces endocytosisof the receptor aggregate and forward movement of the cell (Bristow etal., 2013).

New evidence (unpublished observations) demonstrates that in HIV-1uninfected individuals, CD4+ counts are linearly correlated with thecombination of 4 variables, 1) the number of T-lymphocytes (Ly)expressing HLE-CS and VLDLR, 2) active α1PI levels, 3) inactive α1PIlevels, and 4) T cell antigen receptor rearrangement excision circles(sjβTRECs, a biomarker specific for the generation of newT-lymphocytes). This evidence confirms that α1PI induces generation ofnew CD4+ cells from progenitor cells (Table 2). The mechanism by whichα1PI regulates CD4+ counts is by inducing the migration of hematopoieticprogenitor cells through the thymus (thymopoiesis). As expected, B cells(CD19+) were not found to be correlated with sjβTRECs (r=−0.221,P=0.259, n=28), but were linearly correlated with HLE-CS+ VLDLR+T-lymphocytes, active and inactive α1PI levels (Table 2). Surprisingly,red blood cells (RBC) were also linearly correlated with HLE-CS+ VLDLR+T-lymphocytes, active and inactive α1PI levels (Table 2). Neutrophilsand monocytic cells were not correlated with any of these variables(Table 2). The mechanism by which α1PI regulates CD4+ counts is byinducing the migration of hematopoietic progenitor cells through thethymus (thymopoiesis). As expected, B cells (CD19+) were not found to becorrelated with sjβTRECs (r=−0.221, P=0.259, n=28), but were linearlycorrelated with HLE-CS+ VLDLR+ T-lymphocytes, active and inactive α1PIlevels (Table 2). Surprisingly, red blood cells (RBC) were also linearlycorrelated with HLE-CS+ VLDLR+ T-lymphocytes, active and inactive α1PIlevels (Table 2). Neutrophils and monocytic cells were not correlatedwith any of these variables (Table 2).

Thymopoiesis occurs before birth and continues through adulthood intothe geriatric stage of life when the thymus loses the capacity toproduce new CD4+ T-lymphocytes. Thus, therapeutic α1PI, and pursuant tothe present invention, β-lactams are indicated in the treatment orprophylaxis of secondary immune deficiency when the number of CD4+T-lymphocytes is below normal or predicted to become below normal.Secondary immune deficiencies, also known as acquiredimmunodeficiencies, can result from various immunosuppressive agents.For example, malnutrition, aging and particular medications (e.g.chemotherapy, disease-modifying immunosuppressive drugs administeredafter organ transplants, glucocorticoids). In addition, α1PI, andpursuant to the present invention, β-lactams are similarly indicated inthe treatment of individuals with metabolic syndrome who have elevatedLDL levels.

From the human clinical trial, the following correlations were found:

-   -   1) Increased total cholesterol and LDL were correlated with        increased apoB100 (r=0.82, P=2E-07, n=36 and r=0.89, P=2E-07,        n=36, respectively). This is expected because it is well known        that apoB100 binds to LDL (Veniant et al., 1998);    -   2) Increased triglycerides were correlated with increased apoB48        (r=0.89, P=2E-07, n=36) and increased CD4+ T-lymphocytes, and        the correlation was amplified by α1PI therapy in HIV-1 disease        (r=0.48, P=0.014, n=26), but with decreased monocytes (r=−0.69,        P<2E-07, n=36). This is expected because it is well known that        apoB48 binds to triglycerides and that triglycerides are        primarily transported by CD4+ T cells (Stalenhoef et al., 1984;        Bristow et al., 2013);    -   3) Increased HDL was correlated with increased apoA1 (r=0.82,        P<9.7E-10, n=36); This is expected because it is well known that        apoA1 binds to HDL (Fagerberg et al., 2014).    -   4) Increased total α1PI was correlated with decreased CD3+        T-lymphocytes (r=−0.66, P<1E-06, n=36 and decreased CD8+        T-lymphocytes in which case the correlation was amplified by        α1PI therapy (r=−0.69, P<8E-05, n=25). This is expected because        it was found that increased α1PI is correlated with increased        CD4+ T lymphocytes and increased CD4+ T lymphocytes are        correlated with decreased CD8+ T lymphocytes (Bristow et al.,        2012).        Unexpected results demonstrated the regulation of lipoproteins        by α1PI therapy:    -   1) Increased total α1PI was correlated with decreased apoB48,        and the correlation was amplified by α1PI therapy in HIV-1        disease (r=−0.80, P<2E-06, n=26);    -   2) Increased active α1PI was correlated with decreased HDL        (r=−0.51, P<0.002, n=36);    -   3) Increased HDL was correlated with increased apoA1 (r=0.82,        P<9.7E-10, n=36); This is expected because it is well known that        apoA1 binds to HDL (Fagerberg et al., 2014).    -   4) Increased active α1PI was correlated with decreased apoA1        (r=−0.61, P=7E-05, n=36);    -   5) Increased active α1PI was correlated with decreased        lymphocytes (r=−0.66, P=7.7E-06, n=36);    -   6) Increased inactive α1PI was correlated with increased        lymphocytes, and the correlation was amplified by α1PI therapy        in HIV-1 disease (r=0.42, P=0.037, n=26);    -   7) Increased apoB100 was correlated with increased platelets,        and the correlation was amplified by α1PI therapy in HIV-1        disease (r=0.46, P=0.018, n=26);    -   8) Increased apoB48 was correlated with decreased CD19% (% B        cells) and decreased red blood cells (r=−0.38, P=0.24, n=36 and        r=−0.33, P=0.05, n=36, respectively);    -   9) Increased apoB48 was correlated with decreased total α1PI,        and the correlation was amplified by α1PI therapy in HIV-1        disease (r=−0.80, P=2E-07, n=26);    -   10) Increased total α1PI was correlated with increased apoB100        (r=0.66, P=9E-06, n=36);    -   11) Increased total α1PI was correlated with decreased apoE        (r=−0.56, P=5E-04, n=35);    -   12) Increased total α1PI was correlated with decreased %        CD3+HLE+ T lymphocytes (r=−0.41, P=0.014, n=35) and decreased        CD3+HLE+ T lymphocytes (r=−0, 14, P=0.014, n=35), but not after        treatment;    -   13) Increased total α1PI was correlated with increased sjβTRECs,        a marker of newly generated CD4 T cells (r=0.42, P=0.057, n=21).

These results are unexpected because the relationships between α1PI andlipoproteins other than LDL have not previously been reported. Theseunexpected results demonstrate that triglycerides, using apoB48, aretransported via CD4+ cells from the gut through lymph to blood andfurther, that increased total α1PI decreases CD4+ cells and decreasesapoB48. Interestingly, α1PI therapy in HIV-1 disease amplified thecorrelation between increased total α1PI and decreased apoB48demonstrating that α1PI regulates apoB48 levels and that α1PI and apoB48levels are in feedback regulation in the same manner that α1PI is infeedback regulation with LDL levels (Bristow et al., 2013). These datademonstrate that increased α1PI produces decreased apoB48 therebydecreasing the capacity to absorb dietary fats which results indecreased triglyceride levels. Because LDL levels can be calculatedusing the Friedwald formula as LDL (mg/dL)=total cholesterol (mg/dL)-HDL(mg/dL)-triglycerides (mg/dL)/5, these results show that loweringtriglycerides directly lowers LDL levels. Increased total α1PI wascorrelated with decreased CD3+HLE+ T-lymphocytes (early T cells) andwith increased sjβTRECs (early thymic emigrants) supporting previouslyreports that the generation and fate of T cells are regulated by α1PI.

TABLE 2 Multiple Linear Regression Analysis of Blood Cell Counts inNormal, Healthy Individuals. Independent Variables HLE_(CS) ⁺ VLDLR⁺ LyActive α1PI nactive α1PI sjβTRECs 262 ± 322 events, 22 ± 12 μM, 17 ± 11μM, 403 ± 355 pM, Multilinear Dependent Variables n = 35^(a) n = 36 n =36 n = 28 Regression^(b) CD4 + Ly P = 0.007 P < 0.001 P = 0.006 P =0.034 r² = 0.65, 758 ± 273 cells/μl, P < 0.001, n = 36 n = 27 CD19⁺ Ly P= 0.012 P < 0.001 P < 0.001 NA r² = 0.45, 201 ± 62 cells/μl, P < 0.001,n = 36 n = 35 RBC P = 0.012 P = 0.026 P < 0.001 NA r² = 0.79, 5000 ±500*10⁶ P < 0.001, cells/μl, n = 36 n = 35 Neutrophils P = 0.113 P =0.118 P = 0243  NA r² = 0.10, 3.2 ± 1.3*10⁶ P = 0.352, cells/μl, n = 36n = 35 Monocytic cells P = 0.132 P = 0.200 P = 0.302 NA r² = 0.08, 0.3 ±0.7*10⁶ P = 0.464, cells/μl, n = 36 n = 35 ^(a)Measurements wereobtained from 9 weekly blood samples obtained from 4 normal, healthyindividuals. Values for independent and dependent variables representmean ± standard deviation. Cell counts represent absolute values.HLE_(CS) ⁺VLDLR⁺ cells were quantitated in the CD3⁺CD4⁺ lymphocyte gate(Ly) using flow cytometry. Active and inactive α₁PI levels werequantitated in serum as previously described (Bristow et al., 1998).^(b)Multilinear regression was performed to determine the relationshipof the dependent variables to the independent variables using power oftest α = 0.05. Dependent variables were considered to be significantlyrelated to the independent variable if they contributed significantly tothe multilinear regression (P < 0.05).

Example 8: cDNA Microarray Analysis Demonstrating Feedback RegulationBetween Elastase Inhibitors and Lipoproteins

To examine whether α1PI regulates lipoprotein levels by participating ina regulatory pathway at the cellular level, cDNA microarray analysis wasperformed on two independent primary culture preparations and DNAmicroarray runs. Probe sets ending with x at and s, at were deleted fromanalysis, Monocytic cells (Mo/MO) harvested from 1 uninfected individualand 2 HIV-1 infected individuals on ritonavir therapy were analyzed todetermine the differential expression patterns of 18,400 genes including14,500 functionally characterized genes and 3,900 expressed sequence tagclusters, as previously reported (Modarresi et al., 2009). The data wereobtained using large-scale microarrays performed on two independentprimary culture preparations and DNA microarray runs and the geneexpression ratio of HIV-1 infected to uninfected cells was calculated(Modarresi et al., 2009). All of the genes with lipoprotein andproteinase inhibitor functions that changed more than 2-fold aredepicted. Probe sets ending with x at and s, at were deleted fromanalysis. The results are shown in FIG. 5.

Analysis of gene expression showed that 7 proteinase inhibitors wereupregulated in cells from HIV-1 infected individuals compared to cellsfrom an HIV-1 uninfected control (FIG. 5). Of these, 5 are known to bindto human leukocyte elastase (HLE) including ovalbumin (>254 fold),elafin (>66 fold), skin-derived anti-leukoproteinase (>60 fold),thrombospondin (>50 fold), and α1PI (>17 fold); 1 binds α1PI (heparincofactor, >46 fold); and 1 binds LDL (Tissue Factor PathwayInhibitor, >99 fold). The levels of 4 other proteinase inhibitors weredecreased >12 fold, but none of these inhibitors are known to bind HLE,α1PI, or lipoproteins. LDL receptor (LDLR) and LDL receptor-relatedprotein 5 (LRPS) were increased 4-fold and 8-fold, respectively (datanot shown).

By contrast, the expression of 10 of 12 LDL-binding lipoproteins wasdecreased >2 fold, including scavenger receptor class B (>28 fold),fatty acid binding protein 4 (>19 fold), synaptotagmin III (>18 fold),fatty acid binding protein 7 (>17 fold), lipoprotein Lp(a)-like 2 (>14fold), apolipoprotein L5 (>5 fold), apolipoprotein E (>4 fold),apolipoprotein B mRNA editing protein (>3 fold), and apolipoprotein C-IV(>2 fold) and apolipoprotein L6 (>2 fold). Two lipoproteins wereupregulated, apolipoprotein D (>6 fold) and LDL receptor-related protein5 (>8 fold).

Because α1PI treatment produced decreased LDL in subjects, thismicroarray analysis demonstrates that α1PI is in negative feedbackregulation with LDL and many other lipoproteins.

Example 9: β-Lactam Compounds for Use in the Present Invention

Presented below are compounds which were screened for use in the presentinvention. Two different assays were used as set forth below. In orderto be used in the present invention the compounds must have at least thesame activity as D-ampicillin in both assays.

Assay 1: Inhibition of HLE (microplate assay, 10 tests per plate) asdescribed in Example 2 above and previously published (Bristow et al.,1998). This assay demonstrates the effective binding of the compound tosoluble human leukocyte elastase (HLE-G). D-ampicillin was included as acomparison of potency. Each compound was tested in 2-fold serialdilutions with final concentrations of 0.016 μM to 2 μM versus aconstant concentration of 0.5 μM elastase.

Assay 2: Adherence of cells to compound-coated glass (microwellmicroscope slides, 5 tests per slide) as previously published (Bristowet al., 2008). Adherence is the first step in cell migration andrequires no calcium, magnesium, signaling, or energy. This assaydemonstrates the effective binding of the compound to cell surface humanleukocyte elastase (HLE-CS). As described in Example 1 above, to eachwell of a 10-well microscope slide is added 10 μl of compound at 10-foldserial dilutions with final concentrations of 0.1 nM to 100 nM per well.To each well was added 1×10⁴ cells. After washing and fixing the cells,the number of adherent cells were counted microscopically.

Compounds Tested (n=11):

I. Cephalosporins (Cephems)

-   -   1) Cephalexin (CAS #15686-71-2) (Cayman Chemical 9002009)    -   2) Cefuroxime (CAS #55268-75-2) (American Custom Chemicals Corp.        API0001919)

II. Penicillins (Penams)

-   -   1) Ampicillin (reference activity) (CAS #63-53-4) (American        Custom Chemicals Corp. API0001474)    -   2) Penicillin V (CAS #87-08-1) (American Custom Chemicals Corp.        API0000755)    -   3) Dicloxacillin (CAS #3116-76-5) (American Custom Chemicals        Corp. API0004676)    -   4) Amoxicillin (CAS #34642-77-8) (American Custom Chemicals        Corp. API0015005)    -   5) L-Ampicillin (CAS #19379-33-0)

III. Monobactams

-   -   1) Aztreonam (CAS #78110-38-0) (American Custom Chemicals Corp.        API0001576)    -   2) Ezetimibe (CAS #163222-33-1) (American Custom Chemicals Corp.        API0002672)

IV. Penems

-   -   1) Faropenem (CAS #122547-49-3) (American Custom Chemicals Corp.        API0002676)

V. Carbapenems

-   -   1) Doripenem (CAS #148016-81-3) (American Custom Chemicals Corp.        API0000543)

Example 10: In Vivo Pre-Clinical Study: Effects of L-Ampicillin on TCell Numbers

To examine the ability of L-ampicillin to elevate T lymphocytes, C57BL6mice (6 mice/arm, 2 arms) were administered vehicle control (group 1) orL-ampicillin (5 mg/kg, Group 2). Compounds were delivered by oral gavage5 days per week. Blood was collected weekly for detection of CD3e, CD4,and CD8a T cells by flow cytometry using the mouse T lymphocyte subsetantibody cocktail with isotype control (BD Biosciences). The study wasperformed at the Division of Laboratory Animal Research, Stony BrookUniversity, Stony Brook, N.Y. Staining and statistical analysis wasperformed by Alpha-1 Biologics, and the samples were acquired by theFlow Cytometry Laboratory, Stony Brook Hospital, using a BD LSRFortessainstrument.

Following 2 weeks of treatment with L-ampicillin, as compared withvehicle control, there was a statistically significant increase in CD3+T cells (P<0.05) (FIG. 6A) and CD4+ CD8+ double positive immature,progenitor T cells (P<0.04) (FIG. 6B). Data were normally distributedand compared using Student's T-test. Mean % Change in CD3+ T cells forvehicle was −11% and for CD4+ CD8+ progenitors was −13%. Mean % Changein CD3+ T cells for L-ampicillin (5 mg/kg) was +16% and for CD4+ CD8+progenitors was +38%. Average percent change from baseline isrepresented in FIGS. 6A and 6B where % change=100×[(TreatmentMean-Baseline)/Baseline].

Example 11: L-Ampicillin Treats Solid Tumors in Combination withAnti-PD-1

Tumors persist in the body because the malignant cells are not detectedor targeted by T cells for destruction. The ability of T cells toeffectively target tumor cells is frequently compromised in the tumorenvironment due to the overexpression by tumor cells of molecules thatserve to act as immune checkpoints which are pairs of receptors andligands that moderate and inhibit T cell activity. For this reason,developing immune checkpoint inhibitors has recently become a criticalgoal in cancer therapeutics. For example, programmed cell deathprotein-1 (PD-1) is a receptor on T cells that binds to a ligand (PDL-1)that is overexpressed by tumor cells thereby inhibiting T cell function.Monoclonal antibodies that bind to PD-1 or PD-L1 have been shown to beremarkably effective immune checkpoint inhibitors that reduce tumor sizeand improve prognosis in multiple cancers including melanoma, renal cellcarcinoma, non-small cell lung cancer, bladder cancer, cervical cancer,gastric cancer, liver cancer, pancreatic cancer, and brain cancer. Thetherapeutic use of immune checkpoint inhibitors and the resultingincrease in T cell activity has shown substantial effectiveness toenhance T cell anti-tumor activity and to increase tumor-infiltratingCD4⁺ helper T cells and improve prognosis.

Because L-ampicillin elevates levels of T cells, it was hypothesizedthat L-ampicillin will be effective in treating solid tumors. To testthis hypothesis, BALB/c mice (6 mice/arm, 4 arms) were implantedorthotopically in the left kidney capsule with the syngeneic tumor cellline RENCA (ATCC CRL-2947). Following 1 week of tumor growth, mice weretreated for 3 weeks with anti-PD1 or its isotype control (BioXCell, WestLebanon, N.H., BE0146 and BE0089, respectively) every 4 days IP at asuboptimal dose (7 mg/kg) and L-ampicillin at 5 mg/kg daily by oralgavage. Doses were determined from previously determined in vivo data(Levingston and Young, 2017). The study arms are set forth in Table 3below.

TABLE 3 Dosing Group Dosing Dose Volume # n Test Article Route (mg/kg)(ml/kg) Schedule 1 6 IgG isotype I.P. 7  5 ml/kg Q4D control + P.O. 5 10ml/kg QD L-ampicillin Vehicle 2 6 anti-mouse I.P. 7  5 ml/kg Q4D PD-1 +P.O. 5 10 ml/kg QD L-ampicillin Vehicle 3 6 IgG isotype I.P. 7  5 ml/kgQ4D control + P.O. 5 10 ml/kg QD L-ampicillin 4 6 anti-mouse I.P. 7  5ml/kg Q4D PD-1 + P.O. 5 10 ml/kg QD L-ampicillin

Body weights were obtained 3 times per week to determine treatment dose.Blood was collected weekly for measurement of lymphocyte profile (CD3,CD4, CD8, and immature T cell (CD4+ CD8+ double positives, DPs). On theday of study termination, mice were euthanized and samples werecollected as follows:

-   -   a) Lungs for formalin-fixed, paraffin-embedded (FFPE)        histological staining to detect metastasis of tumor cells;    -   b) Left and right kidney weight;    -   c) Tumor physical appearance and size.

There were detectable tumors in untreated mice, mice treated withanti-PD-1, and mice treated with L-ampicillin (FIGS. 7A and 7B). Incontrast, there were no detectable tumors in any of the 6 mice treatedwith the combination of anti-PD-1 and L-ampicillin. There were noadverse effects detected from treatment.

Excised kidneys were visually examined and photographed. Representativetumors are depicted in FIG. 7A. Mean tumor weight within each group wasdetermined as the difference between the left kidney weight and theright kidney weight in each mouse (FIG. 7B).

Example 12: Physical Properties

D-ampicillin (CAS no. 69-53-4) and L-ampicillin (CAS no. 19379-33-0) arediastereomers. In biological systems, drugs that are diastereomersexhibit different chemical reactions, e.g., D-ampicillin is soluble inethanol whereas L-ampicillin is not.

The absolute configuration of D-ampicillin and L-ampicillin are depictedin FIGS. 8A and 8B, respectively.

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The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all values are approximate, and areprovided for description.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

What is claimed is:
 1. A method for increasing the number of CD4+T-lymphocytes in the serum of a subject in need of such treatmentcomprising administering to the subject a pharmaceutical compositioncomprising an amount of the L-isomer of a β-lactam effective to increasethe number of CD4+ T-lymphocytes in said patient's serum wherein saidβ-lactam has substantially no bactericidal activity and wherein theβ-lactam is a tromethamine salt of L-ampicillin.
 2. The method of claim1 further comprising the step of determining the number of CD4+T-lymphocytes in said patient's serum before administration of thepharmaceutical composition.
 3. The method of claim 1 further comprisingthe step of determining the number of CD4+ T-lymphocytes in saidpatient's serum after administration of said pharmaceutical composition.4. The method of claim 1 wherein said subject is infected with HIV-1. 5.The method of claim 1 wherein said subject is suffering from secondaryimmune deficiency.
 6. The method of claim 1 wherein said subject issuffering from inherited α1PI deficiency.
 7. The method of claim 4,wherein the number of CD4+ T-lymphocytes in said subject's serum isbelow normal.
 8. The method of claim 1 wherein said subject is a patientexposed to environmental toxins or undergoing cancer therapy.
 9. Themethod of claim 1 wherein said subject is suffering from at least onecondition selected from the group consisting of viral infection,bacterial infection, and malnutrition.
 10. The method of claim 4,wherein said subject is receiving HIV anti-retroviral therapy.
 11. Themethod of claim 1 wherein said β-lactam is a member selected from thegroup consisting of Cephems, Monobactams, Penams, Penems andCarbapenems.
 12. The method of claim 1 further comprising administeringa pharmaceutically acceptable carrier or diluent.
 13. The method ofclaim 8, wherein said cancer therapy further comprises administering acheckpoint inhibitor with said L-isomer of a β-lactam.
 14. The method ofclaim 13 wherein said checkpoint inhibitor is a monoclonal antibody thatbinds programmed cell death protein-1 (PD-1).
 15. The method of claim 8wherein said cancer is a solid tumor.
 16. The method of claim 15 whereinsaid cancer is a member selected from the group consisting of melanoma,renal cell carcinoma, non-small cell lung cancer, bladder cancer,cervical cancer, gastric cancer, liver cancer, pancreatic cancer, andbrain cancer.